The overall goal of this competing NIH R01 renewal application is to create a tumor-targeted cell-penetrating peptide (CPP) nanoscale drug delivery system to target therapeutic payloads to a wide range of solid tumors. This proposal builds upon the observation that oligoarginine peptides display a strong threshold effect in their cell penetration ability; below a threshold of 6 consecutive arginine (Arg) residues in the CPP, little cell uptake is observed, while above this threshold number of Arg residues, there is significant cell uptake. We hypothesized that this threshold effect is not related to the number of sequential Arg residues in the oligoarginine CPP, but instead is reflective of the local Arg residue density. This hypothesis predicts that the triggered self-assembly of a diblock copolymer, that presents < 6 Arg residues on its hydrophilic end, into a micelle should provide a system that exhibits digital off-on cell uptake. To test this hypothesis and develop an externally triggered CPP drug delivery system, thermally responsive diblock copolymers of an elastin-like polypeptide (ELPBCs) will be synthesized such that the number of Arg residues on the hydrophilic terminus of the ELPBC will be below the threshold required for efficient cellular uptake of ELPBC unimers at 37 C. In tumors that are externally heated to 39-42 C, which is greater than the critical micellization temperature of the ELPBC, the ELPBC will self-assemble into micelles decorated with Arg residues, and the local Arg residue density in the corona of the ELPBC will exceed the threshold required for efficient cellular uptake, thereby resulting in efficient intracellular uptae within the tumor. Arg-presenting ELPBC with a range of architectural variables will be synthesized, tested for self-assembly and stability between 39 and 42 C in serum, and their temperature-triggered cellular uptake will be quantified by flow cytometry and visualized by fluorescence microscopy. Optimal Arg-presenting ELPBC selected from these studies will then be tested for their pharmacokinetics, tissue distribution and their ability to regress tumors, when they are fused to gemcitabine that is sequestered within the core of the ELPBC micelles. The significance of this proposal is that it will provide a receptor independent method of actively targeting tumors that is applicable to a broad range of cancer types and circumvents the limitations of receptor targeting; furthermore this targeting modality piggybacks on to existing hyperthermia technology, so that it can be readily deployed in the clinic. The drug carrier design presented here is innovative because it is, to our knowledge, the first attempt to control cellular uptake of cancer therapeutics by manipulating the local density of Arg residues on a nanoscale scaffold by externally triggered self-assembly under clinically relevant conditions.